The cytoskeleton constitutes a large family of proteins that are involved in many critical processes of biology, such as chromosome and cell division, cell motility and intracellular transport. Vale and Kreis (1993) Guidebook to the Cytoskeletal and Motor Proteins New York: Oxford University Press; Alberts et al. (1994) Molecular Biology of the Cell, 788-858). Cytoskeletal proteins are found in all cells and are involved in the pathogenesis of a large range of clinical diseases. The cytoskeleton includes a collection of polymer proteins, microtubules, actin, intermediate filaments, and septins, as well as a wide variety of proteins that bind to these polymers (polymer-interacting proteins). Some of the polymer-interacting proteins are molecular motors (myosins, kinesins, dyneins) (Goldstein (1993) Ann. Rev. Genetics 27: 319-351; Mooseker and Cheney (1995) Ann. Rev. Cell Biol. 111: 633-675) that are essential for transporting material within cells (e.g., chromosomal movement during metaphase), for muscle contraction, and for cell migration. Other groups of proteins (e.g., vinculin, talin and alpha-actinin) link different filaments, connect the cytoskeleton to the plasma membrane, control the assembly and disassembly of the cytoskeletal polymers, and moderate the organization of the polymers within cells.
Given the central role of the cytoskeleton in cell division, cell migration, inflammation, and fungal/parasitic life cycles, it is a fertile system for drug discovery. Although much is known about the molecular and structural properties of cytoskeletal components, relatively little is known about how to efficiently manipulate cytoskeletal structure and function. Such manipulation requires the discovery and development of specific compounds that can predictably and safely alter cytoskeletal structure and function. However, at present, drug targets in the cytoskeleton have been relatively untapped. Previous studies have been directed towards drugs that interact with the cytoskeletal polymers themselves (e.g., taxol and vincristine), and towards motility assays. Turner et al. (1996) Anal. Biochem. 242 (1): 20-5; Gittes et al. (1996) Biophys. J. 70 (1): 418-29; Shirakawa et al. (1995) J. Exp. Biol. 198: 1809-15; Winkelmann et al. (1995) Biophys. J. 68: 2444-53; Winkelmann et al. (1995) Biophys. J. 68: 72S. In general, the studies on polymerization and motility were preliminary research studies performed in an effort to define the existing mechanisms of these actions. Although the cytoskeletal system has been characterized to some extent, studies have not focused on the binding interactions of the polymers such as microtubules and actin with various polymer binding proteins such as molecular motors with a specific goal of identifying and characterizing modulators of such interactions that could have biopharmaceutical and bioagricultural relevance. In particular, there is still a need in the art to identify compounds which can be used to manipulate the cytoskeletal system, particularly in regards to modifying the binding characteristics of the cytoskeletal components to one another. In particular, there is a need in the art to identify compounds that modulate the cytoskeletal binding interactions which can be used as therapeutics and/or diagnostics, as well as compounds which can be used in the bioagriculture field (e.g. as pesticides). It is noted that virtually no effort has been directed to finding agents (e.g. drugs) that target cytoskeletal proteins that bind to the different filaments and which are expected to provide targets of greater specificity and thereby provide fewer unwanted side effects when targeted with various modulators.
The invention herein provides convenient and rapid methods for identifying compounds not previously known to modulate cytoskeletal function. In particular, this invention provides assay methods that include methods for measuring binding interactions between cytoskeletal polymers and cytoskeletal polymer-binding proteins that can be applied to high-throughput screening to identify small molecules that modify this interaction. The methods described herein include methods that provide high sensitivity and can be used in complex mixtures, including, but not limited to crude cell extracts.